Click-chemistry-based protocol to quantitatively assess fatty acid uptake by Mycobacterium tuberculosis in axenic culture and inside mouse macrophages

Summary Mycobacterium tuberculosis (Mtb) hijacks host-derived fatty acids (FAs) to sustain its intracellular growth inside host cells. Here, we present a click-chemistry-based protocol to assess FA import by Mtb in axenic culture or inside mouse macrophages. We describe the use of alkyne analogs of natural FAs as an alternative to structurally altered fluorescent derivatives or hazardous radiolabeled FAs. We also detail quantitative analyses of FA uptake at single bacterial or host cell level by flow cytometry and confocal fluorescence microscopy. For complete details on the use and execution of this protocol, please refer to Laval et al. (2021).1


SUMMARY
Mycobacterium tuberculosis (Mtb) hijacks host-derived fatty acids (FAs) to sustain its intracellular growth inside host cells. Here, we present a click-chemistry-based protocol to assess FA import by Mtb in axenic culture or inside mouse macrophages. We describe the use of alkyne analogs of natural FAs as an alternative to structurally altered fluorescent derivatives or hazardous radiolabeled FAs. We also detail quantitative analyses of FA uptake at single bacterial or host cell level by flow cytometry and confocal fluorescence microscopy. For complete details on the use and execution of this protocol, please refer to . 1

BEFORE YOU BEGIN
As an intracellular pathogen, Mycobacterium tuberculosis (Mtb) relies on host-derived lipids including fatty acids (FAs) for survival and growth in vivo. 2 Mtb's ability to import FAs in axenic cultures or inside macrophages was previously studied using radiolabeled or fluorophore-conjugated FAs, two approaches with advantages and disadvantages. [3][4][5] While radiolabeled FAs are structurally identical to the native compounds, they require an appropriate infrastructure and are not compatible with single cell analyses. Conversely, fluorescent derivatives of FAs allow such analyses but the covalent addition of relatively large fluorophores greatly alters their structure and physicochemical properties. This may impact their import and use by bacteria and eukaryotic cells, as reported for a commonly-used fluorescent derivative of glucose. [6][7][8] Here, we describe a protocol adapted from the one previously published by Nazarova and colleagues and using a BODIPY-conjugated FA. 9 This new version uses commercially available alkyne-FAs that are structurally close to the native FAs and can be detected by a highly specific and versatile 'click' reaction (copper(I)-catalyzed alkyne-azide cycloaddition) with a picolyl azidetagged fluorophore. 10 The steps below describe the various applications of this protocol, using the example of Mtb strain H37Rv grown in axenic culture or internalized by murine bone marrowderived macrophages (BMDMs). We have also used this protocol successfully with M. bovis BCG and human PMA-differentiated THP-1 macrophages.
All media and solutions must be prepared before experiments (see recipes in the materials and equipment section below). They can be stored as indicated in the footnotes of the recipes.

Institutional permissions
All animal procedures were performed in agreement with European and French guidelines (Directive 86/609/CEE and Decree 87-848 of 19 October 1987). The study received the approval by the Institut Pasteur Safety Committee (Protocol 11.245).

Preparation of L929-conditioned medium
Timing: 7 days 1. Grow L929 cells from frozen stocks, and split the culture to plate 1 3 10 6 cells in 100 mL of complete DMEM in a TC-treated 150-cm 2 culture flask. 2. Incubate cells for 7 days at 37 C and 5% CO 2 . 3. Harvest the cell culture medium.
a. Centrifuge the pooled L929-conditioned medium at 200 3 g for 5 min at 22 C. b. Pool all supernatants and filter them through a sterile 0.22 mm filter. c. Aliquot in 50 mL tubes and store at À20 C for up to a year.
To take into account variations in the concentration of M-CSF secreted by L929, a quality check of each new batch of conditioned medium (ability to promote BMDM differentiation, see section below) is recommended.

Preparation of bone marrow-derived macrophages (BMDMs)
Timing: 7 days 4. For microscopy experiments, prepare a 24-well plate containing sterile coverslips. a. Autoclave glass coverslips and tweezers with a dry cycle. b. In a biosafety cabinet, place coverslips in pure ethanol in a Petri dish. c. Crook a sterile, beveled needle with autoclaved tweezers. Using tweezers and the crooked needle, transfer coverslips in the wells of the plate. d. Let the ethanol evaporate for 1 h in the biosafety cabinet. e. Wash the wells containing the coverslips once with 1 mL of sterile 13 DPBS before seeding the cells. 5. Euthanize a C57BL/6J mouse using the approved methods of primary and secondary euthanasia at your institution. Use 70% ethanol to clean the exterior of the animal. 6. In a biosafety cabinet, dissect out femur and tibia bones and place them in 13 DPBS as described previously by Toda and colleagues. 11 7. Soak the bones in 70% ethanol for 1 min, transfer in new sterile 13 DPBS, cut off extremities and flush them with 13 DPBS using a syringe and a 25G needle as described. 11 8. Homogenize the cell suspension by pipetting up and down, and pass it through a 70-mm cell strainer on top of a sterile 50 mL conical tube. 9. To count the isolated hematopoietic cells, dilute 10 mL of cell suspension in 90 mL of Tü rk's solution, and use a Malassez counting chamber. 10. Seed 7 3 10 6 cells in 10 mL of BMDM differentiation medium per 100 mm TC-treated cell culture dish, or 2 3 10 5 cells in 500 mL medium per well of a 24-well plate containing autoclaved coverslips. 11. Culture bone marrow cells for 6 days, with medium change on day 3. 12. After 6 days of differentiation, replace BMDM differentiation medium with BMDM culture medium and incubate another 24 h at 37 C and 5% CO 2 .
Note: Using this method, 60-80 million bone marrow cells can reproducibly be isolated from one 7-12 week-old C57BL/6J male mouse.
Note: The quality of BMDM differentiation can be assessed by flow cytometry as described previously by Toda and colleagues. 11 A 90% rate of CD11b-and F4/80-positive cells is expected with this protocol.
Note: Alternative protocols have been described for the generation of BMDMs. We have not tested if the outcome of the assays described below is different with these alternative protocols. Any alterations of BMDM lipid metabolism (specifically their FA import capacity) could have significant effects on the assay outcome. This protocol was adapted from that previously reported for the uptake of radiolabeled FAs by Mtb grown in axenic culture. 3,4 The variation presented below uses flow cytometry to assess quantitatively the uptake of alkyne-FAs at the single-bacterial level in vitro. 3. Alkyne FA uptake: a. To make a heat-killed negative control, incubate an aliquot of the culture for 15 min at 80 C. b. Let it cool down to 22 C-25 C before incubation with alkyne FAs.

KEY RESOURCES
Note: An alternative is to kill bacteria by treatment with 4% PFA for 30 min at 22 C-25 C, before centrifugation (9,000 3 g for 5 min) and resuspension in alkyne-FA-supplemented 7H9-AD.   5. Wash bacterial pellets once in ice-cold 7H9-AD medium. 6. Wash bacterial pellets twice in ice-cold Wash Buffer.
Note: These washing steps were optimized to minimize the amount of FAs that are bound to the mycobacterial envelope without being internalized, using heat-killed Mtb as a metabolically inert control. Note: Since BMDMs are not replated after differentiation in this protocol, we use an approximative multiplicity of infection (MOI) of 2 bacilli per cell, assuming that an OD 600 of 1 corresponds to 1.5 3 10 8 bacterial/mL and that the BMDM monolayer corresponds to 7 3 10 6 cells in 100 mm dish and 2 3 10 5 cells/well in 24-well plate. CRITICAL: For confocal microscopy analysis, proceed to click chemistry staining as described in the next section. For flow cytometry analysis of FA uptake by Mtb, intracellular bacteria have to be isolated from infected BMDMs prior to staining. 18. After the FA uptake assay described above, wash twice with ice-cold 13  Note: The copper:protectant ratio (see materials and equipment section) in the cocktail was optimized for this staining to minimize the deleterious effects of copper on GFP fluorescence while conserving a maximal reaction efficiency (as indicated by the clicked FA signal in bacteria incubated with alkyne FAs), as per the manufacturer's indications (https://www. thermofisher.com/order/catalog/product/C10643).
Note: Although we optimized this click reaction to be compatible with GFP-expressing Mtb, we noticed that copper has much less deleterious effects on the fluorescence of RFP-expressing Mtb. Therefore, the amount of copper may be adjusted when using strains expressing other fluorescent proteins. Imaging software, or equivalent. 33. For each acquisition, set up the z-scan spanning so that most intracellular bacteria are fully imaged. 34. For quantification of FA uptake, perform all acquisitions using the same settings.
a. Define Mtb and BMDM regions of interest (ROIs) by the GFP and clicked FA signal, respectively, using the HK-Means plugin 16 of the Icy opensource platform 12 (see Figure 1). b. Measure the mean fluorescence intensity in defined ROIs.

Timing: 2-3 h
This protocol uses the Click-iT TM Plus Alexa Fluorä 647 Picolyl Azide Toolkit to assess and quantify alkyne-FA import by Mtb from axenic culture or infected macrophages.
Note: This speed is necessary to pellet fixed bacteria in a buffer devoid of detergent (otherwise, bacteria tend to stick to the walls of plastic tubes).  Figure 2). Exclude small debris by selecting GFP-positive events in the FITC channel. 49. Analyze the AF647 fluorescence signal in the APC channel, using GFP-expressing bacteria isolated from infected macrophages not treated with alkyne-FAs as negative control.
Note: There is not requirement for compensation when using GFP and AF647, since they have negligible spectrum overlap.
Note: Care should be taken to assess FA uptake by host macrophages during infection, as it determines FA bioavailability for intracellular Mtb. 1 FA uptake by infected macrophages can easily be assessed in parallel by confocal microscopy as described above, or by staining unlysed cells (following the Click-IT TM toolkit manufacturer's instruction (https://www.thermo fisher.com/order/catalog/product/C10643) and flow cytometry analysis.
Note: For assays in axenic cultures, we do not completely eliminate bacterial clumps that may form throughout the protocol, but simply use the 35-mm cell strainer to avoid large clump formation. However, when a very heterogenous population is observed in flow cytometry analyses (indicative of large clumps), alkyne-FA signals are normalized by FSC values for each event using the ''Derive Parameters'' function in FlowJo.

EXPECTED OUTCOMES
FA uptake in axenic culture Using this approach, we confirmed that the uptake of alkyne-FAs is severely impaired in Mtb strains deficient for Mce1, the only specific FA transport system characterized to date in Mtb. 17 Importantly, we compared FA uptake by live versus heat-or PFA-killed Mtb. As shown in Figure 3A, both killing methods strikingly reduced FA uptake by Mtb, confirming that our assay largely measures active import of FAs. Of note, in killed bacteria we could detect a residual FA signal that is likely corresponding to passive diffusion of alkyne FAs.
To validate that the alkyne modification did not significantly alter the mechanism and efficiency of FA import by Mtb, we performed competition assays with increasing doses of natural FAs added to a fixed dose of the alkyne FA counterpart. As expected, the uptake of all alkyne-FAs was efficiently competed by addition of their natural counterpart ( Figure 3B).

FA uptake inside macrophages
We were able to detect a significant defect in the import of several alkyne-FAs in Mce1-deficient Mtb (Dmce1D) using both confocal microscopy ( Figures 4A and 4B) and flow cytometry ( Figure 4C). The

Potential solution
For axenic cultures, make sure that all Mtb strains were at the same growth stage. Although we were able to measure significant differences after 1 h of FA uptake and have not tested other timepoints for these comparisons, bigger differences may be observed with longer uptake period. If the problem persists, bacteria should be washed more thoroughly before fixation (assay of fatty acid uptake by Mtb in axenic cultures, steps 5 and 6 & assay of fatty acid uptake by Mtb inside BMDMs, steps 17.k-q).

Problem 2
The signal of the fluorescent protein expressed by the Mtb strain used is very low in click stained compared to unstained samples.

Potential solution
This signal depends on the copper sensitivity and expression levels (controlled by the strength of the promoter used) of the fluorescent protein in your Mtb strains. The copper:protectant ratio in the Click-iT TM Plus reaction cocktail (materials and equipment (optional), click chemistry staining for analysis by confocal microscopy, step 24) may have to be adjusted to limit the deleterious effects of copper on the fluorescent protein.

Problem 3
There is minimal competition between an alkyne-FA and its natural counterpart for uptake by Mtb in axenic culture.

Potential solution
This may be caused by a lack of stability or solubility of the compounds. Make sure you keep the ethanol stock solutions of FAs as cold and away from light sources as possible while preparing the pre-conjugated FA-BSA (materials and equipment (optional)). In case of doubt, replace the stock solution by an unopened one (unsaturated FAs are particularly sensitive to oxidation). To ensure good solubility in the culture medium, mix thoroughly FAs in pre-warmed FA-free BSA and incubate for a minimum of 20 min at 37 C (this time can be increased, but without exceeding 1 h to limit the oxidation of unsaturated FAs).

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources should be directed to Caroline Demangel (demangel@pasteur.fr).

Materials availability
The M. tuberculosis strains used in this protocol may be made available upon request.

Data and code availability
This study did not generate/analyze datasets or code.

ACKNOWLEDGMENTS
This study was supported by Fondation pour la Recherche Mé dicale (T.L.; FDT201904008040) and core funding from Institut Pasteur (T.L., C.D.) and INSERM (U1224, C.D.). We thank the Image Analysis Hub of Institut Pasteur for confocal microscopy data analysis. We also thank Prof. Luke Chamberlain (University of Strathclyde, Glasgow, UK) for sharing his expertise of FA labelling by click chemistry.